This is generally in the area of compositions for treatment of cancer, in particular, compositions containing blockers of the protein C system in combination with a lymphokine.
A variety of mechanisms in tumors capable of promoting clot formation have been described (Dvorak, H. F. Human Path. 18,275-284 (1987); Rickles, F. R., Hancock, W. W., Edwards, R. L., et al. Sem.Thromb.Hemost. 14,88-94 (1988)). Initially, the discovery of extravascular fibrin deposits in a variety of animal and human tumors prompted the search for these tumor-associated clotting mechanisms. This extravascular fibrin disposition has been found in association with prothrombin, factor VII and factor X in certain tumor cells in situ by immunohistochemical techniques. A heat- and acid-stable glycoprotein present in mucin produced by certain adenocarcinomas is capable of catalyzing the conversion of factor X to factor Xa. A 68,000 dalton cysteine protease has been identified in a number of tumor lines which activates factor X, independent of the actions of factor VIIa and tissue factor. Some tumor homogenates display tissue factor activity, while other tumor cell lines have cell membranes with receptors for factor Va and are capable of catalyzing the conversion of prothrombin to thrombin. Abnormalities in coagulation parameters observed in certain cancer patients has prompted the hypothesis that tumor-associated clotting may be of sufficient magnitude to cause systemic activation of the clotting system.
Alterations in the fibrinolytic system are observed in tumors and transformed cells. Plasminogen activator activity has been found to be higher in extracts of surgically excised human cancer tissues than in surrounding benign tissue. In addition to other possible roles for plasminogen activator, such as participation in tumor invasion of normal tissue, this suggests that tumors have the capacity to promote fibrin degradation. Urokinase-type plasminogen activator appears to be produced by tumors with greater frequency than tissue-type plasminogen activator. It is not known if the production of plasminogen activators by tumors is involved in preventing thrombus accumulation within the bed of the tumor.
Protein C is a vitamin K-dependent plasma protein. Activated protein C serves as a natural anticoagulant by inhibiting the clotting cascade at the levels of factor Va and factor VIIIa (Walker, F. J., Sexton, P. W. and Esmon, C. T. Biochim.Biophys.Acta 571,333-342 (1979); Fletcher, C. A., Gardiner, J. E., Griffin, J. H., et al. Blood 63,86-49 (1984)). Protein C is rapidly converted to activated protein C by a complex of thrombin and the endothelial cell surface protein, thrombomodulin. Thrombomodulin forms a 1:1 stoichiometric complex with thrombin and increases the rate at which thrombin activates protein C by approximately 20,000-fold. This activation occurs primarily in the capillaries, where the availability of a large endothelial surface area per unit of plasma volume favors complex formation between thrombin and thrombomodulin. The activation of protein C in the microvasculature forms a potential feedback loop which, when thrombin is formed in the circulation, generates activated protein C. The activated protein C in turn inhibits further thrombin formation. This has been demonstrated directly in dogs, where low level intravenous thrombin infusion results in the generation of activated protein C and anticoagulation of the animal. A more complete review of the roles of thrombomodulin and protein C in regulation of blood coagulation is by C. T. Esmon, in J. Biol. Chem. 264(9), 4743-4746 (1989).
Thrombomodulin has been identified on a variety of cultured endothelial cell lines and on the luminal surface of blood vessels (Esmon, N. L. Semin.Thromb.Hemost. 13,454-463 (1987)). Thrombomodulin has also been identified by functional and immunochemical means on tumor cells, including human lung carcinoma -line CL-185 and Bowes melanoma cells (Marks, C. A., Bank, N. U., Mattler, L. E., et al. Thromb.Hemost. 54,119 (1985)), A549 human lung cancer cells (Maruyama, I. and Majerus, P. W. Blood 69,1481-144 (1987)), and angiosarcomas (Yonezawa, S., Maruyama, I., Sakae, K., et al. Am.J.Clin.Pathol. 88,405-11 (1987)). The functional role of thrombomodulin on the tumor cells is subject to speculation. Tumor associated thrombomodulin may help to protect the tumor from excess fibrin formation. If the protein C-thrombomodulin system is involved in determining the hemostatic balance in the tumor vasculature, blocking protein C activation may shift the hemostatic balance and result in thrombosis of tumor vessels.
In addition to functioning as an anticoagulant, activated protein C promotes fibrinolysis. This action involves complex formation between activated protein C and two inhibitors of plasminogen activator, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 3 (PAI-3). Complex formation between activated protein C and PAI-1 or PAI-3 may serve to protect plasminogen activators from inhibition and can thus potentially result in an increase in fibrinolytic activity. The extent to which in vivo protein C influences fibrinolytic activity in the body or in tumor beds in particular is unknown.
Protein S, another vitamin K-dependent plasma protein, serves as a cofactor for the anticoagulant and fibrinolytic effects of activated protein C (Walker, F. J. Semin. Thromb. Hemost. 10,131-138 (1984); de Fouw, N. J., Haverkate, F., Bertina, R. M., et al. Blood 67, 1189-1192 (1986). Protein S exists in two forms in plasma. Forty percent of the protein S is free and serves as a cofactor for activated protein C, and 60% is in complex with C4b binding protein and is functionally inactive. C4b binding protein is an acute phase protein (Boerger, L. M., Morris, P. C., Thurnau, G. R., et al. Blood 69,692-694 (1987); Dahlblack, B. J.Biol.Chem. 261,12022-12027 (1986)) and thus inflammation, by elevating the levels of C4 binding protein, may shift protein S to the inactive form by the law of mass action. This shift in protein S status may predispose to thrombosis. Hereditary protein S deficiency, at least in some kindreds, is linked to an increased risk of venous thrombosis. See, for example, P. C. Comp, et al., J. Clin. Invest. 74, 2082-2088 (1984).
While heterozygous protein C deficiency in certain kindreds is also associated with an increased risk of venous thrombosis, two other protein C deficiency states are characterized by tissue necrosis: homozygous protein C deficiency and the coumarin-induced skin necrosis observed in heterozygous protein C deficient individuals after the initiation of coumarin therapy. In homozygous deficient individuals, skin necrosis occurs on the first or second day of life, resulting in a clinical condition termed purpura fulminans neonatalis. Thrombosis of the small vessels of the skin is characteristic, leading to loss of large areas of skin, which is often fatal. Major vessel thrombosis is also possible.
When oral anticoagulant therapy is initiated in heterozygous protein C deficient patients, extensive microvascular thrombosis may occur. The skin is again the primary target and extensive loss of skin and underlying tissue can occur. The postulated mechanism is a rapid fall in protein C levels in the heterozygous deficient individuals before the levels of clotting factors with a long half-life, such as prothrombin and factor IX, decrease to levels adequate for systemic anticoagulation. A transient hypercoagulable state may exist in these individuals and it is during this period that the tissue necrosis occurs.
Heterozygous protein C deficiency is relatively common in the population and may occur as frequently as 1 in 300 individuals. Coumarin necrosis is rare and this suggests that factors other than protein C deficiency alone must be present. A review of the literature indicates that most patients developing necrosis have some inflammatory condition as well such as an infection, recent surgery or extensive venous thrombi. These accompanying inflammatory changes could decrease thrombomodulin expression, increase tissue factor expression and favor a shift of free protein S to the inactive C4b binding protein-protein S complex. These events would further down regulate the protein C system and promote microvascular clot formation, suggesting that protein C deficiency accompanied by inflammation can result in tissue necrosis.
Although spontaneous regression of solid tumors can occur on rare occasions following febrile illnesses, Dr. William B. Coley demonstrated in Annals of Surgery 14,199-220 (1891) that regression of certain solid tumors in humans could follow the administration of heat killed bacteria. The response of the tumors was highly variable and the diagnosis of the tumor type was not always made by histologic examination. However, some of the patients treated with Coley""s toxins had long term tumor regression and possible cure. Coley""s work and that of other investigators testing the effects of bacterial products on animal tumors, led to the discovery of tumor necrosis factor, a 157 amino acid molecule capable of causing tumor necrosis in a number of murine tumors (Old, L. J. Nature 330,602-603 (1987); Old, L. J. Scientific American 258,59-75 (1988); Gifford, G. E. and Flick, D. A. Tumor Necrosis Factor and Related Cytotoxins, edited by Bock, G. and Marsh, J. Chilchester, p. 3-20 (John Wiley and Sons, 1987). Tumor necrosis factor is produced by macrophages in response to endotoxin. Tumor necrosis factor triggers a number of physiologic responses on skeletal muscle, adipose tissue, endothelium, cartilage, leukocytes and the hypothalamus, and has a direct cytotoxic effect on some tumor cell lines (Tracey, K. J., Lowry, S. F. and Cerami, A. Tumour Necrosis Factor and Related Cytokines, p. 88-108 (1987).
Tumor necrosis factor causes inflammatory changes at the endothelial level by increasing the adhesion of PMNs. blood monocytes and related leukocyte cell lines. This results from the tumor necrosis factor induced production of endothelial-leukocyte adhesion molecules (E-LAMs) by the endothelium (Pober, J. S., Bevilacqua, M. P., Mendrick, D. L., et al. J.Immunol. 136,1680-1687 (1986); Pober, J. S., Lapierre, L. A., Stolpen, A. H., et al. J.Immunol. 138,3319-3324 (1987); Bevilacqua, M. P. and Gimbrone Jr., M. A. Sem.Thromb.Hemost. 13,425-433 (1987). Tumor necrosis factor also stimulates endothelial cell production of platelet activating factor which may also promote microvascular thrombosis by platelet activation and activation of adherent polymorphonuclear leukocytes (Pober, J. S. Tumour Necrosis Factor and Related Cytokines p. 170-184 (1987).
The effects of tumor necrosis factor on the endothelium include increased tissue factor activity and decreased thrombomodulin expression (Nawroth, P. P. and Stern, D. M., J. Exp. Med. 163, 740-745 (1986)). Tumor necrosis factor and interleukin-1 can decrease the production of human cultured umbilical vein endothelial cells tissue-type plasminogen activator and increase the production of plasminogen activator inhibitor type 1 (Schleef, R. R., Bevilacqua, M. P., Sawdey, Mr., et al. J.Biol.Chem. 263,5797-5803 (1988); Nachman, R. L., Hajjar, K. A., Silverstein, R. L., et al. J.Exp.Med. 163,1595-1600 (1986); Emeis, J. J. and Kooistra, T. J.Exp.Med. 163,1260-1266 (1980); Bevilacqua, M. P., Schleef, R. R., Gimbrone, M. A., Jr., et al. J.Clin.Invest. 78,587-591 (1986). These findings, coupled with the microvascular thrombosis observed in certain protein C deficiency states, suggest that simultaneous administration of tumor necrosis factor and inhibition of the protein C system in vivo may result in intravascular thrombosis.
There is evidence that activated protein C influences the production of TNF in the intact animal. The administration of activated protein C protects against shock induced by the infusion of E. coli, as described in U.S. patent application Ser. No. 07/139,922 entitled xe2x80x9cTreatment of Dysfunctional Vascular Endothelium Using Activated Protein Cxe2x80x9d filed Dec. 31, 1987 by Fletcher B. Taylor Jr and Charles T. Esmon, now issued as U. S. Pat. No. 5,009,889. In the animals receiving activated protein C, the production of tumor necrosis factor in response to the E. coli is markedly reduced, raising the possibility that blockade of protein C activation in vivo could result in enhanced production of TNF by macrophages or natural killer cells in the tumor bed in the presence of low levels of endotoxin. This could in turn contribute to further toxic effects on the tumor.
Palladino, et al., in J.Immunol. 138,4023-4032 (1987); Tumour Necrosis Factor and Related Cytokines, p. 21-38 (1987); has proposed that injection of tumor necrosis factor into Meth A sarcoma bearing mice actually causes a series of events which result in tumor rejection: 1) hemorrhagic tumor necrosis, initiated in the first one to four hours involving PMN activation and their localization to the tumor; 2) direct cytostatic/cytotoxic effects on the tumor as growth stops, at 24 to 72 hours; and 3) a specific T-cell mediated immune response to the tumor, at two to four weeks. Blockade of the protein C system may potentiate certain aspects of the TNF mediated necrosis in addition to promoting microvascular thrombosis. Blockade of the protein C system should result in increased thrombin generation by tissue factor at the endothelial surface. The thrombin which is produced can increase endothelial cell production of platelet activating factor, which would prime marginated granulocytes and thus enhance the PMN mediated endothelial cell injury.
Anticoagulants block the Shwartzman reaction, presumably by preventing fibrin deposition and microvascular thrombosis (Edwards, R. L. and Rickles, F. R. Science 200,541-543 (1978). If tumor killing by tumor necrosis factor does have characteristics in common with the Shwartzman reaction, blocking protein C activation could allow intravascular thrombosis to proceed unimpeded and increase the extent and severity of damage to the microvasculature, and thus increase tumor killing. Since the use of tumor necrosis factor in the treatment of human malignancies is accompanied by serious side effects, protein C blockade, which may potentiate the tumor-directed effects of a given dose of tumor necrosis factor, should be worth investigation.
It is therefore an object of the present invention to provide a method and compositions to block the natural anticoagulant pathways, to thereby promote microvascular coagulation in the new capillaries growing into tumors and by so doing block the process of angiogenesis.
It is a further object of the present invention to provide a method and compositions to promote an immune response against tumors by blocking the natural anticoagulant pathways.
Compositions, and methods of use thereof, for the inhibition of tumor growth and killing of solid tumors wherein the active agent is a compound blocking the Protein C system, preferably anti-Protein C or anti-activated Protein C antibody, anti-Protein S antibody, inactivated activated Protein C, and C4b binding protein, administered systemically or at the site of the tumor. In the most preferred embodiment, the Protein C blocking compound is provided in combination with a cytokine that stimulates natural killer and lymphokine-activated killer cell-mediated cytotoxicity, activates macrophages, stimulates Fc receptor expression on mononuclear cells and antibody-dependent cellular cytotoxicity, enhances HLA class II antigen expression, and/or stimulates procoagulant activity, such as tumor necrosis factor (TNF), gamma interferon, granulocyte-macrophage colony stimulating factor, interleukin-1, interleukin-2, or endotoxin, which elicits production of cytokines such as TNF. It is possible to stop or reverse the hypercoagulapathy following administration of the Protein C blocking compound by administration of an agent inhibiting the blocking compound, such as by administration of Protein C, or more preferably activated Protein C, when the blocking compound is anti-Protein C antibody. Although initial studies show that a single treatment is effective in significantly reducing tumor size, different Protein C blocking compounds, alone or in combination with different cytokines and/or other anti-tumor agents, such as chemotherapeutic agents or anti-tumor monoclonal antibodies, can be administered simultaneously or subsequently to the initial treatment to reduce tumor size. Radiation and hyperthermia can also be used to sensitize the tumor to the protein C blocking agent.
Examples are provided demonstrating the systemic and local administration of the Protein C blocking compound, alone or in combination with TNF, to dogs having canine venereal tumors, a fibrosarcoma, and an adenocarcinoma, and to pigs having melanomas, followed by significant tumor reduction. Systemic administration of a second anti-Protein C antibody, alone or in combination with LPS (endotoxin) to mice was also effective in causing regression of Meth A sarcomas in mice models.